(a) Utilization of Silica as Chromatographic Support
Silica in a variety of particulate forms has been extensively utilized as a chromatographic support. Partition of dissolved molecules between the hydrophilic siliceous surface and a flowing solvent permits the separation of compounds on many different scales (ng→kg scales). The efficiency of separation in these systems is related to the surface area of the silica to which the compound mixture is exposed.
The configuration of common separation systems utilizes a cylindrical bed of particulate silica in a glass, metal or polymeric cladding. A traditional approach to improving separation efficiency (theoretical plates) with such systems is to utilize longer columns of particulate silica of a given particle size (or range of sizes). Alternatively, higher separation efficiency is associated with the use of very small particles with larger surface areas.
There is an important physical limitation to practical separation with packed particulate systems. As the number of theoretical plates increases there is an attendant increase in backpressure on the column. There is, therefore, a trade off between higher separation efficiency and practical operating pressures. High pressures have attendant danger, and/or are impractical from the perspective of cost. Even with highly efficient columns operating at high pressures, the throughput that can be realized is often relatively low.1 
Significant improvement in the surface area/back pressure relationship can be realized by the use of self-supporting monolithic silica columns.1,2 For example, styrene monoliths have been reported to be useful for polynucleotide separation.3 The group of Tanaka, in particular, have reported the preparation of silica monoliths.4 Merck currently sells monolithic silica columns under the Chromolith” label.5 The structure of these monoliths involves a series of distorted silica spheres fused by a layer of silica. The presence of macropores, between linked silica beads of a few microns diameter, can be clearly seen by micrographic analysis and may be more carefully established by other techniques. In addition to macropores, the silica beads typically possess a mesoporous structure (in the case of the Merck columns, a total porosity of 80% is claimed, which is made up of macro- and mesopores, the latter of which are on the order of 13 nm in diameter).5 
(b) Problems with Existing Monolithic Silica
Silica produced by a sol-gel process is prone to shrinkage. Gelation is initiated in the presence of large quantities of solvent and, frequently, other dopants (see below). Evaporation of the solvent is accompanied by significant shrinkage forces: Si(OEt)4-derived gels can shrink in air up to 85%.6 This can be problematic in a number of ways when the resulting silica is used as a chromatographic support. First, in extreme conditions, the column can fracture leading to changes/degradation in separation performance. Second, the monolith can pull away from the cladding material, providing an alternative elution pathway for the compounds to be separated. This complicates, at best, the separation. In the worst instance, the eluting mixture will bypass most of the column surface area resulting in no separation.
Several strategies have been developed to reduce the problem of shrinking. For example, use of a drying agent in the original sol, such as DMF, helps in the silica annealing process.7 The most common means to deal with shrinking is to accept that it will occur and to thermally cure the silica, essentially to completion. Hydrothermal treatment can be used to dissolve/re-precipitate the silica, which reduces the cracking that is frequently observed upon shrinking.8,9 Dopants like urea in the sol have been reported to facilitate the dissolution/re-precipitation process.10 An alternative strategy is to heat shrink the column cladding after shrinkage has occurred to reform an effective interface between monolith and cladding material. Finally, soluble polymers such as poly(ethylene oxide) may be added to the sol. These have the effect of increasing porosity of the monolith.11 
The use of sol-gel techniques provides an exceptional degree of morphological control in the preparation of silica. Thus, total porosity, pore size and shape, regularity of pore distribution, etc., can be manipulated using a variety of starting materials, reaction conditions and dopants.12 Many of these conditions, however, are incompatible with the incorporation of fragile compounds such as biomolecules, proteins in particular. Either the synthetic conditions are damaging to protein structure (e.g., pH conditions, the presence of denaturants such as ethanol) or the final curing conditions require elevated temperatures. It is of interest to incorporate such biomolecules into silica to create materials that serve as biosensors, immobilized enzymes or as affinity chromatography supports.
(c) Applications of Monolithic Silicas to Bioaffinity Chromatography
Bioaffinity chromatography has been used widely for sample purification and cleanup,13 chiral separations,14 on-line proteolytic digestion of proteins,15 development of supported biocatalysts,16 and more recently for screening of compound libraries via the frontal affinity chromatography method.17,18 In all cases, the predominant method used to prepare protein-loaded columns has been based on covalent or affinity coupling of proteins to silica beads. However, coupling of proteins to beads has several limitations, including; loss of activity upon coupling (due to poor control over protein orientation and conformation), low surface area, potentially high backpressure (which may alter Kd values19), difficulty in loading of beads into narrow bore columns, difficulty in miniaturizing to very narrow columns (<50 μm i.d.), and poor versatility, particularly when membrane-bound proteins are used.18 
In recent years it has been shown that a very mild and biocompatible sol-gel processing method can be used to entrap active proteins within a porous, inorganic silicate matrix.20 In this method, a two-step processing method is used wherein a buffered solution containing the protein is added to the hydrolyzed silica sol to initiate gelation under conditions that are protein-compatible.21 Numerous reports have appeared describing both fundamental aspects of entrapped proteins, such as their conformation,22,23,24 dynamics,25,26,27 accessibility,24,28 reaction kinetics,22,29 activity,30 and stability,31 and their many applications for catalysis and biosensing.20,21 A number of reports also exist describing sol-gel based immunoaffinity columns,32 and enzyme-based columns33 although in all cases these were formed by crushing protein-doped silica monoliths and then loading the bioglass into a column as a slurry.
Very recent work on the development of protein-doped monolithic sol-gel columns has appeared from the groups headed by Zusman34 and Toyo oka.35 Zusman s group have developed columns using glass fibers covered with sol-gel glass as a new support for affinity chromatography. Toyo oka s group have used capillary electrochromatography (CEC) to both prepare protein-doped sol-gel based columns and to elute compounds. These monoliths were derived solely from TEOS or TMOS using a very high water:silicon ratio, resulting in a loosely packed monolith with large pores to allow flow of eluent. While this is a significant advance, all chromatography was done using electroosmotic flow (CEC), which separates compounds on the basis of a combination of charge, mass and affinity, and is less compatible with MS detection due to the necessarily high ionic strength of the eluent. Also, these authors did not examine the interaction of potential inhibitors with entrapped proteins on-column. This is a particularly important issue given the emergence of high throughput screening (HTS) methods based on immobilized enzymes.17,18,36 
The present inventors have previously described the preparation of silica from a series of sugar alcohol, sugar acid or oligo- and polysaccharide-derived silanes. These starting materials offer a number of advantages over the more classically used tetraethoxy- and tetramethoxysilanes (TEOS and TMOS, respectively). Among these are mild conditions, including a greater control of pH used in the sol (ranges from 4-11.5 are possible), very low processing temperatures, process reproducibility, reduced shrinking and compatibility with the incorporation of a variety of dopants, particularly proteins. However, there remains a need to control the shrinkage of the resulting silica to avoid the evolution of cracks. Furthermore, morphological control needs to be available such that the materials can be tailored for specific applications including chromatography, biosensors, etc. Finally, an ability to improve the stability of the entrapped biomolecule is needed.